A novel fluorescence-activated cell sorting (FACS)-based screening identified ATG14, the gene required for pexophagy in the methylotrophic yeast

Abstract Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.


Introduction
Autophagy is an intracellular degradation system that is highly conserved among eukaryotes.In yeast species, autophagy can be classified as macr oautopha gy or micr oautopha gy.In macr oautopha gy, autopha gosomes separ ated b y bilay ers sequester the cytoplasmic cargo and fuse with the vacuole for degradation (Suzuki et al. 2001 , Suzuki andOhsumi 2007 ).Micr oautopha gy, in contr ast, involv es the dir ect engulfment of the tar get car go by the v acuole.During the process, the vacuolar membrane randomly invaginates and differentiates into the autophagy tube (Wang et al. 2022 ).Autophagy pathways can be further divided into nonselective or selecti ve types.Nonselecti ve autophagy involves the random delivery of a portion of the cytoplasm to the v acuole, wher eas selectiv e autopha gy r ecognizes and degr ades specific car goes , e .g. organelles, a ggr egates, and pathogens (Lamark and Johansen 2021 ).These conserv ed pr ocesses ar e ac hie v ed thr ough the coordination of different autophagy-related (ATG) proteins and more than 40 ATG proteins have been discovered to date (Ohsumi 2014 ).
Pexophagy is a selective autophagy pathway for the specific degradation of peroxisomes.Based on the vacuolar membrane dynamics, pexophagy is categorized into macr opexopha gy and micr opexopha gy (Oku and Sakai 2018 ).In the methylotrophic yeast K omagataella phaffii (syno ym Pic hia pastoris ), macr opexopha gy is in-duced upon the carbon source shift from methanol to ethanol.Macr opexopha gy belongs to the macr oautopha gic pr ocess in whic h individual per oxisomes ar e sequester ed by ne wl y synthesized membrane structures called pexophagosomes (Tuttle and Dunn 1995 ).Micr opexopha gy, on the other hand, is induced after the medium tr ansition fr om methanol to glucose.During the pr ocess, per oxisomes ar e dir ectl y engulfed by v acuolar sequestering membranes (VSMs) extending from the septated vacuole and a double-membr ane structur e called micr opexopha gyspecific membr ane a ppar atus (MIPA) (Mukaiyama et al. 2004 ).The formation of both MIPA and pexophagosomes is mediated by ATG proteins (Oku and Sakai 2016 ).To date, several ATG proteins requir ed for pexopha gy hav e been identified by plate-based assays performed primarily under micropexophagy-inducing conditions (Tuttle and Dunn 1995, Sakai et al. 1998b, Mukaiyama et al. 2002, Dunn et al. 2005 ) as screening under macropexophagy-induced conditions has often yielded false positive results.
In recent years, fluorescence-activated cell sorting (FACS) has become a po w erful method for screening specific gene mutants fr om lar ge libr aries.It is a specialized instrument that can query m ultiple fluor escence par ameters of individual particles suc h as cells and microbeads at a rate of ∼10 7 per hour (Shapiro 2005 ).Taking full adv anta ge of its high-thr oughput natur e, the a pplication of FACS scr eening has been expanded to various research fields including autophagy (Morita et al. 2018, Shoemaker et al. 2019 ).
In this study, we established a nov el high-thr oughput FACSbased screening method to identify the genes r equir ed for macr opexopha gy.Thr ough the screening, we discov er ed the KpATG14 gene, which could not be identified for a long time due to technical limitations of existing methods.KpAtg14 sho w ed relativ el y low similarity to ScAtg14 in the budding yeast Saccharomyces cerevisiae and SpAtg14 in the fission yeast Schizosaccharom yces pombe , wher eas critical domains suc h as the cysteine r epeats and coiled-coil domains at the N-terminus were conserved.Thr ough micr oscopic and imm unoblot anal yses we r e v ealed that KpAtg14 was r equir ed for both macr o-and micr opexopha gy.In addition, we also found that KpAtg14 was necessary for recruiting downstr eam ATG pr oteins, and thus for bulk autopha gy degr adation, in a manner similar to that of ScAtg14.

Yeast strain, culture conditions, and reagents
The yeast strains used in this work are listed in Table 1 .Cells wer e gr own at 28ºC on the a ppr opriate media described below.Yeast extract, peptone, and dextrose (YPD) medium consisted of 1% yeast extract, 2% bactopeptone, and 2% glucose; synthetic dextrose (SD) medium contained 0.67% yeast nitrogen base without amino acids and 2% glucose; synthetic methanol (SM) medium had 0.67% yeast nitrogen base without amino acids and 0.7% methanol; synthetic ethanol (SE) medium consisted of 0.67% yeast nitrogen base without amino acids and 0.5% ethanol; and synthetic dextrose without nitrogen source (SD-N) medium contained 0.67% yeast nitrogen base without amino acids and ammonium sulfate and 2% glucose.Yeast extract, yeast nitrogen base without amino acids and dextrose (YND), yeast extract, yeast nitrogen base without amino acids and methanol (YNM) and yeast extr act, yeast nitr ogen base without amino acids and ethanol (YNE) media were made with the addition of 0.5% yeast extract to SD, SM, and SE media, r espectiv el y.These synthetic media wer e supplemented with the a ppr opriate amino acids (100 μg/ml arginine and/or 100 μg/ml histidine) and/or antibiotics (100 μg/ml zeocin), as necessary.The pH of SD, SM, SE, YND, YNM, and YNE media was adjusted to 6.0 with NaOH.Gro wth w as monitored b y measuring the optical density at 610 nm (OD 610 ).The nucleotide sequence of KpATG14 was deposited in the DDBJ/EMBL/GenBank under accession number LC815816.
The vector used for FACS-based screening was constructed as follows: The primer pair pHluorin-infusion_Fw/pHluorin-infusion_Rv was used to amplify the fluorescent protein pHluorin-coding sequence using pBW1679 as template .T he PCR-amplified fr a gment was then fused with Spe I/ Hind III cut fr a gment of pIB1, using In-Fusion Cloning Kit (Clontec h), whic h resulted in pMN001.Then, the KpPex11 promoter and the ORF region without the STOP codon were amplified using primers P ex11-pHluorin-infusion_Fw/P ex11-pHluorin-infusion_Rv using the genomic DNA as template .T he obtained fr a gment was fused with Eco RI/ Spe I cut fr a gment of pMN001 using In-Fusion Cloning Kit (Clontec h), whic h r esulted in pMN002.Finall y, the primer pairs P11-pH_in v_linker_fw/P11-pH_in v_linker_rv and mC_in v_linker_fw/mC_in v_linker_rv were used to linearize pMN002 and amplify the fluor escent pr otein mCherry-coding sequence from pmCherry-C1 as a template , respectively.T hese fr a gments wer e fused with eac h other, r esulting in the v ector pMN003 used for FACS-based screening.
A deletion cassette for the KpATG14 gene was constructed as follows: Primer pair A TG14UP_Fw1/A TG14DOWN_Rv1 was used to amplify a 3.1-kb fr a gment using the genomic DNA as template .T he fr a gment, including KpATG14 ORF and its upstr eam and downstr eam r egions, was cloned into pIB1 between Eco RI and Hind III sites using In-Fusion Cloning Kit (Clontec h), r esulting in pTN001.Then, KpATG14 ORF, amplified by the primer pair A TG14_HIS_Fw/A TG14_HIS_Rv, w as replaced b y the zeocin resistance gene that was amplified from SK + Zeo r using the primer pair Zeo_Fw/Zeo_Rv, yielding the KpATG14 disruption vector pTN002.To disrupt the KpATG14 gene, the disruption cassette was PCR-amplified from the disruption vector, pTN001, using the primer pair A TG14d_Fw/A TG14d_Fw, and transformed into K. phaffii PPY12 by electr opor ation.Pr oper gene disruptions were confirmed by colony PCR.
The vectors harboring tagged KpATG14 were constructed as follows: the KpATG14 promoter and ORF r egion wer e PCR-amplified using primers ATG14UP_Fw2 and ATG14DOWN_Rv2 and using the genomic DNA as template .T he fr a gment was fused to pIB1, which was linearized by PCR with the primers pIb1-Arg_Fw and pIb1-Ar g_Rv, r esulting in pARM001.The fluorescence protein Ceruleancoding sequence was amplified from pRSET A -Redoxfluor-C-probe as a template with primers CeruleanORF_Fw/CeruleanORF_Rv and inserted into pARM001, which was linearized by PCR with the primers pIb1-Ar g_ATG14_Fw/pIb1-Ar g_ATG14_Rv, r esulting in pARM002.The infusion reaction to construct these vectors was performed with In-Fusion Cloning Kit (Clontech).For the construction of pARM003 and pARM004, pARM001 was linearized by PCR with the primer pairs A TG14-FLAG_Fw/A TG14-FLAG_Rv and A TG14-FLAG C84_F/A TG14 C84-FLAG_Rv, r espectiv el y, and the PCR products were subjected to self-ligation.
The vector encoding KpIDH1-YFP expressed under its native promoter was constructed by swapping GFP coding sequence with YFP coding sequence .T he v ector pIB1-IDH1-GFP, used pr e viousl y (Yamashita et al. 2016 ), was linearized by restriction enzymes Kpn I and Bam HI.The fr a gment YFP coding sequence was PCR amplified with a primer set YFP_KpnI_Fw/YFP_BamHI_Rev using pNT812 as a template, and treated with restriction enzymes Kpn I and Bam HI.These two fr a gments wer e ligated with Ligation-Conv enience Kit (NIPPON GENE), r esulting in pKS0001.The v ector encoding KpATG5-CERULEAN expressed under its native promoter was constructed as follows: the KpATG5 promoter and ORF region without the STOP codon were amplified using primers A TG5_infusion_Fw/A TG5_infusion_Rv using the genomic DNA as template .T he obtained fr a gment was inserted into pARM002, which was linearized by PCR with primers pIb1-Ar g_Rv/ATG14 C84-Ceruean_Fw, r esulting in pARM005.The vector encoding KpATG6-3xHA expressed under its native promoter was constructed as follows: The KpATG6 promoter and ORF region without the STOP codon were amplified using primers

Restriction enzyme-mediated integration mutagenesis
Restriction enzyme-mediated integration (REMI) was used to facilitate a r andom m utation of genes in the K. phaffii genome by the incor por ation of a zeocin-r esistance gene.Tr ansformation of K. phaffii with a pREMI-Z vector was carried out after linearizing it with Bam HI.Transformed cells were selected b y gro wth on zeocin plates .T hose transformed cells unable to degrade peroxisomes during ethanol adaptation were identified through the two-step (FACS and fluor escence micr oscopy) scr eening, described below.Basic methods used for the identification of the genes r equir ed for macr opexopha gy wer e described pr e viousl y (Sc hr oder et al. 2007 ).To find mutation sites, sequencing analyses were performed with the primer set pREMIZ_plus_f3 and pREMIZ_plus_r4.

Pexophagy induction
Macr opexopha gy for the FACS based screening was induced as follows.A single colony was inoculated onto YPD medium and cultiv ated ov ernight.A volume of 40 μl of cells in 5 ml of YPD wer e tr ansferr ed to 5 ml of fr esh YPD medium and incubated for 5 h as a pr ecultur e.To pr epar e the cell samples used for the first scr eening with FACS, pr ecultur ed cells wer e gr own in 5 ml of SM medium for 12-16 h, tr ansferr ed to 5 ml of SE medium and incubated for 6 h to induce macr opexopha gy.For the second screening with fluorescence microscope, cells plated on a membrane (Amersham HybondTM-N + , GE Healthcare) were incubated on SM agar medium for 18 h.Then, the membrane was tr ansferr ed onto SE agar medium and incubated for 4 h to induce macropexophagy.Macr opexopha gy and micr opexopha gy in inv estigating the function of KpAtg14 were induced as follows.A single colony was inoculated onto YPD medium and cultivated o vernight.T he cells wer e tr ansferr ed to 5 ml of YNM medium containing 0.93 μg/ml FM4-64 (Invitr ogen), whic h labeled the v acuolar membr ane .T he cells were incubated in 5 ml of YNM medium for 12-16 h and were shifted to 5 ml of YNE medium (macropexophagy) or YND medium (micr opexopha gy) and examined.

Bulk autophagy and mitophagy induction
A single colony was inoculated into YPD medium and cultivated overnight.A volume of 40 μl of cells in 5 ml of YPD were transferred to 5 ml of fresh YPD medium and incubated for 5 h as a pr ecultur e .T hen, the pr ecultur ed cells wer e tr ansferr ed to SD-N medium.

FACS
Cells wer e r esuspended in ice-cold phosphate-buffer ed saline buffer and k e pt on ice.Flow cytometry was performed using FACSAria III (Becton Dic kinson).Fluor escent c hannel and light scatter were set at log gain.The forw ar d scatter (FSC) w as set at a photomultiplier tube (PMT) voltage of 23 with a threshold of 800.The PMT voltages of side scatter (SSC) and green fluor escent pr otein (GFP) wer e set at 225 and 500, r espectiv el y. pHluorin fluorescence was excited with a 488-nm laser, and the emission at 530/30 nm was detected.mCherry fluorescence was Table 2. Primers used in this study.

DNA sequence
Resulting plasmids CGGA GTCCGA GAAAATCTGG excited with a 561-nm laser, and the emission at 610/20 nm was detected.At least 50 000 cells were analyzed per sample .T heoreticall y, KpPEX11-pr omoter-dependent fluor escence of pHluorin and mCherry is detected when cells are grown on methanol, showing the correlated dot plots of the cell population both in pexophagyacti ve and -defecti ve cells.After the carbon source shifts to ethanol, the fluorescence of pHluorin is decreased in pexophagyactive cells, while the fluorescence of mCherry is maintained.On the other hand, the fluorescence of both pHluorin and mCherry are maintained in pexophagy-defective cells .T herefore , when the dot plots of pexopha gy-activ e and -defectiv e cells ar e mer ged, pexopha gy-activ e cell populations can be observed.As such, cells were gated for FSC and SSC to select single cells, as described in Fig. 1 (C).FACSDiva 8 software (Becton Dickinson) was used for data acquisition and creation of scatter plots.Bioconductor ( www.bioconductor.org ) on the open-source statistical platform R ( www.r-pr oject.org ) was used for processing data, follo w ed b y the pr epar ation of histogr ams.Candidates of pexopha gy m utants were sorted and transferred into 5 ml of YPD medium.After harv est, cells wer e plated onto YPD a gar medium and incubated for 2-3 days until colonies a ppear ed.T hese colonies , candidates of pexopha gy m utants, wer e used for the second scr eening with fluor escence micr oscopy.

Fluorescence microscopy
Cells were observed using an IX81 fluorescence microscope (Olympus) equipped with a XF52 filter set (Omega Optical Inc.) for FM4-64 and fluorescent proteins.Image data were captured with a SenSysTM c har ged-coupled de vice camer a (PhotoMetrics) and analyzed on MetaMorph imaging software (Universal Imaging Cor por ation), Ima geJ2 v ersion 2.3.0 and Adobe Illustrator.In all figures, the scale bar was set to 2 μm.

Morphometric analysis
Cell count analysis was performed in cells observed under fluorescence microscopy ( n > 30, n ; number of cells analyzed and f > 3, f ; a field of vision).Independent examinations were repeated at least three times.

Prepar a tion of protein extracts from yeast cells
The samples were prepared from cells harvested at an OD 610 of 2.0.The cells were resuspended in 1 ml of solution I that contained 0.2 M NaOH and 0.5% (v/v) 2-mercaptoethanol.Then the cell samples were incubated on ice for 10 min and 0.1 ml of 100% (w/v) tric hlor oacetic acid solution was added.The cell l ysates wer e subjected to centrifugation at 20 000 g at 4 • C for 5 min.After the This study supernatant was r emov ed, the pellet was resuspended in 1 ml of acetone by brief sonication.The obtained sample was then subjected to centrifugation at 20 000 g at 4 • C for 5 min and the pellets were dried.Subsequently, the pellets were dissolved in 80 μl of sample buffer, containing 0.1 M Tris-HCl (pH 7.5), 2% (w/v) SDS, 1% (v/v) gl ycer ol, 0.5% (v/v) 2-mercaptoethanol, and 0.01% (w/v) bromophenol blue, by brief sonication.The samples were then incubated at 65 • C for 10 min, follo w ed b y centrifugation at 20 000 g for 1 min.A 20-μl volume of the supernatant was electr ophor esed on a 10% SDS-PAGE gel.

Immunoprecipitation
Komagataella phaffii cells were collected, resuspended in HNE buffer (25 mM HEPES-KOH pH 7.2, 150 mM NaCl, and 2 mM EDTA), and ruptured using Multi-Beads Shocker (Yasui Kikai) at 2500 rpm for 30 s with 0.5-mm YZB zirconia beads (Yasui Kikai).Subsequently, the same volume of HNE buffer containing 0.2% dodecylβ-D-maltoside (DDM, from Dojindo Molecular Technologies) was added to the lysates, incubated for 20 min on ice, and then centrifuged at 10 000 rpm for 15 min.The supernatants were incubated with anti-FLAG magnetic beads (Wako Pure Chemical) with gentle rotation for 3 h.The beads were then washed three times with the HNE buffer containing 0.03% DDM and the bound proteins were eluted with SDS-PAGE sample buffer.

Results
The FACS-based screening identified the genes required for macr opexopha gy The yeast cells, expressing a peroxisomal protein KpPex11 (Oku and Sakai 2016 ) fused with a tandem-tagged fluorescent protein pHluorin-mCherry, were used for FACS-based screening.In principle, when KpPex11-pHluorin-mCherry encounters the vacuole via the pexophagic process, the fluorescence signal of the pH-sensitiv e GFP v ariant pHluorin is r educed under the internal low pH conditions, while mCherry exhibited diffused fluorescence, which could be sorted through the screening (Fig. 1 A).To examine the methodological validity, we first observed the wildtype (WT) strain and Kpatg30 strain, a complete pexophagydefectiv e m utant (Farré et al. 2008, Ohsawa et al. 2021 ), expressing KpP ex11-pHluorin-mCherry b y a fluor escence micr oscope during macr opexopha gy-induced conditions.As expected, upon the induction of macr opexopha gy, r eduction of pHluorin fluorescence and diffusion of mCherry fluorescence were observed in the WT strain, whereas in the Kpatg30 strain both pHlluorin and mCherry fluor escence wer e clearl y detected under macr opexopha gy-induced conditions (Fig. 1 B).Next, we analyzed the WT and Kpatg30 strains by flow cytometry during the shift of carbon source from methanol to ethanol, inducing macr opexopha gy.When these str ains wer e gr own on methanol, an identical plot pattern of cell populations was obtained, with the fluorescence intensity of pHlluorin and mCherry showing a correlation (Fig. 1 C).After the shift to ethanol-containing medium, on the other hand, the WT strain sho w ed more cell populations emitting weaker fluorescence intensity of pHlluorin than the Kpatg30 strain.In particular, in the area indicated by the red circle in the enlar ged ima ge, onl y four plots a ppear ed in the WT str ain, wher eas 5764 plots were present in the Kpatg30 strain (Fig. 1 C), suggesting that the flo w c ytometry analysis can distinguish WT cells from Kpatg30 cells and that the highlighted area can be used as a sorting gate for screening macropexophagy mutants.
Using the WT strain expressing KpP ex11-pHluorin-mCherry, w e designed an experimental protocol to identify the genes required for macr opexopha gy, whic h entailed a two-step scr eening pr ocesses by FACS and fluor escence micr oscop y (Fig. 1 D).Accor dingly, r andom m uta genesis was performed and ∼50 000 transformants wer e obtained.Tr ansformants wer e then scr eened using FACS by selecting the cells sorted by the gate shown in Fig. 1 (C), which yielded 227 candidates for the second screening (data not shown).The candidate transformants were then spotted on a membrane, which was placed on a methanol-containing medium and transferred to an ethanol-containing medium, inducing macropexopha gy.Fluor escence micr oscopy anal ysis narr o w ed the list of candidates down to 84 (data not shown).Subsequently, sequencing analysis was performed to find mutation sites in the 84 transformants and 9 different genes that had mutations in their ORF region were identified (Table 4 ).Among the nine genes, we found one unknown gene that contained a 1584-bp ORF encoding a protein of 528 amino acids .T he other eight genes, namely ATG1 , ATG7 , A TG9 , A TG11 , A TG12 , A TG18 , A TG24 , and A TG26 (Yuan et al. 1999, Kim et al. 2001, Strømhaug et al. 2001, Mukaiyama et al. 2002, 2004, Oku et al. 2003, Stasyk et al. 2003, Ano et al. 2005 ), have alr eady been discov er ed and c har acterized by pr e vious studies.A PSI-BLAST search (Jones and Swindells 2002 ) at the NCBI database found that the amino acid sequence of the unknown gene had a weak but significant homology to ScAtg14 in S. cerevisiae (Identity: 14%, Similarity: 27%) ( Fig. S1 ).Ther efor e, we named the putative gene KpATG14 and performed the following experiments.

Char acteriza tion of KpAtg14
We first asked whether KpAtg14 contained conserved amino acid sequences found among yeasts .T he PSI-BLAST search with KpAtg14 identified a putative OpAtg14 in another methylotrophic yeast Ogataella polymorpha together with well-c har acterized ScAtg14 in S. cerevisiae and SpAtg14 in S. pombe.Alignment analysis of KpAtg14 sho w ed r elativ el y low homologies to SpAtg14 (identity: 13%, similarity: 39%) and OpAtg14 (identity: 18%, similarity: 54%), similar to ScAtg14 ( Fig. S1 ).Investigating the amino acid sequences, we found that KpAtg14, as well as OpAtg14, contained a conserved sequence motif of cysteine repeat at the N terminal region ( Fig. S1 ).In addition, the coiled-coil prediction (Simm et al. 2015 ) indicated that KpAtg14 and OpAtg14 possessed a conserved sequence motif of a coiled-coil domain at the N terminal region ( Fig. S2 ).These results indicated that KpAtg14 has conserved motifs found among yeasts (Fig. 2 A).

Functional analysis shows that KpAtg14 is required for macr opexopha gy
The KpATG14 gene was disrupted by replacing the ORF with the zeocin resistance gene as a selective marker.We then inv estigated the r ole of KpAtg14 in macr opexopha gy by performing the morphological analysis in the Kpatg14 strain.A fluor escent pr otein CFP fused with the per oxisomal tar geting signal 1 (PTS1) and YFP-tagged KpAtg8 were used as markers of per oxisomes and pexopha gosomes, r espectiv el y (Mukaiyama et al. 2004 ).The vacuolar membranes were stained with FM4-64.YFP-KpAtg8 was observed as dot-like structures both in the WT and Kpatg14 strains (Fig. 2 B).Ho w ever, clear CFP diffusion in the vacuole was observed only in the WT strain 2 h after the medium shift (Fig. 2 B and C).In the Kpatg14 strain, CFP fluor escence r emained by the side of the v acuole, suggesting that the Kpatg14 strain is a complete macr opexopha gy-defectiv e m utant.Macr opexopha gic activity was also investigated by immunoblot analysis using cells expressing KpPex11 fused with a fluorescent protein pHluorin, as described previously (Yamashita et al. 2017 ).Per oxisome degr adation was confirmed by detecting the cleav a ge of KpPex11-pHluorin in the WT strain.On the other hand, the cleaved form of pHluorin was detected in neither the Kpatg14 nor the Kpatg30 strain (Fig. 2 D and E), which was consistent with the microscopic analyses .Furthermore , we examined whether Cerulean-tagged KpAtg14 colocalizes with YFP-KpAtg8 under macr opexopha gy-induced conditions.Upon the induction of macr opexopha gy, KpAtg14-Cerulean was observ ed to colocalize with YFP-KpAtg8 (Fig. 2 F).Taken together, we concluded that KpAtg14 was essential for macr opexopha gy in K. phaffii .

KpAtg14 is also required for micr opexopha gy
Next, we asked whether KpAtg14 is essential for micr opexopha gy.Mor phological anal ysis was performed in the WT and Kpatg14 strains, in a manner similar to the macr opexopha gic observ ation described abo ve .In the WT strain, a cluster of peroxisomes started to be enwr a pped by the fr a gmented v acuoles with the association of YFP-KpAtg8 1 h after the medium shift and a diffusion of the CFP fluorescence was detected in the following hours, indicating the occurrence of micr opexopha gy (Fig. 3 A and  B).On the other hand, in the Kpatg14 strain, although peroxisomes wer e enwr a pped by the fr a gmented v acuoles in a similar UDP-glucose:ster ol glucosyl-tr ansfer ase Oku et al. ( 2003 ), Stasyk et al. ( 2003 ) manner to the WT strain, CFP diffusion was not observed (Fig. 3 A  and B).Detailed morphometric analysis with the yeast cells 1 h after the macr opexopha gy induction confirmed that the same le v el of VSMs budding from the vacuole was detected in the WT and Kpatg14 strains (Fig. 3 C), suggesting that KpAtg14, together with other ATG proteins , pla ys an indispensable role in the final stage of micropexophagy such as MIPA formation for the complete enclosure of peroxisomes in the vacuole (Mukaiyama et al. 2002 ).Our pr e vious study pr oposed a model of micr opexophagy in K. phaffii , in which micropexophagy is divided into four distinct mor phological sta ges, termed sta ge 0 to stage 3 (Sakai et al. 1998b ).Considering that per oxisomes wer e enwr a pped by VSM in a similar manner to the WT strain, the phenotype of the deletion of KpATG14 could be categorized into stage 1c in which most ATG mutants such as Kpatg1 and Kpatg7 are grouped.Furthermor e, imm unoblot anal ysis with the WT, Kpatg14 , and Kpatg30 strains found that micropexophagic activity was defective in the Kpatg14 and Kpatg30 strains (Fig. 3 D and E).We also examined the intracellular localization of KpAtg14-Cerulean and YFP-KpAtg8, a marker protein of MIPA under macropexophagyinduced conditions (Mukaiyama et al. 2004 ).Upon the induction of micr opexopha gy, KpAtg14-Cerulean was observ ed to colocalize with YFP-KpAtg8 (Fig. 3 F).These results indicated that KpAtg14 is r equir ed for micr opexopha gy in K. phaffii .

KpAtg14 is dispensable for mitophagy
Subsequentl y, we inv estigated the effects of deletion of ATG14 on mitopha gy, another selectiv e autopha gy pathway for mitoc hondria.To monitor the mitophagic activity, the yeast strains expr essing a mitoc hondrial matrix pr otein KpIdh1 fused with YFP were constructed (Yamashita et al. 2016 ).The cells cultured on a nutrient-rich medium were transferred to nitrogen-starvation conditions and collected over time for immunoblot analysis .T he cleaved form of YFP was clearly detected in the WT strain during starvation-induced mitophagy (Fig. 4 A and B).In the Kpatg14 str ain, mitopha gy was slightl y impair ed but activ e, indicating that KpAtg14 is not r equir ed for starvation-induced mitophagy.Considering that ScAtg14 is indispensable for mitophagy in the budding yeast S. cerevisiae (Yamashita et al. 2016 ), K. phaffii may have differ ent r egulation mec hanism for mitopha gy.

KpAtg14 is required for nonselecti v e bulk autophagy
We then examined whether KpAtg14 is r equir ed for nonselective bulk autophagy with the yeast strains expressing the cytoplasmic protein KpPgk1 fused with CFP, as pr e viousl y described (Pang et al. 2019 ).When the cells were subjected to nitr ogen-starv ation conditions , the clea v ed form of CFP was clearl y detected in the WT strain (Fig. 5 A and B).On the other hand, the deletion of the ATG14 gene se v er el y impeded bulk autophagic degradation under nitr ogen starv ation.We also inv estigated the intr acellular localization of KpAtg14.When cells were cultured on YPD medium, KpAtg14-Cerulean was diffused in the cytosol.Upon the induction of bulk autophagy, KpAtg14-Cerulean colocalized with YFP-KpAtg8, whic h was observ ed as foci (Fig. 5 C).These r esults indicated that KpAtg14 functions at the pr eautopha gosomal structur e (PAS) and is necessary for bulk autophagy.We further examined whether KpAtg14 is necessary for the recruitment of downstr eam ATG pr oteins for autophagosome formation.In S. cerevisiae , ScAtg5, which is required for lipidation of ScAtg8, functions downstream of ScAtg14 in the autophagosome formation, and deletion of the ScATG14 gene causes a drastic decrease in the localization of ScAtg5 at the PAS (Suzuki et al. 2007 ).Upon the induction of bulk autophagy, the puncta of YFP-KpAtg8 were detected in both the WT and Kpatg14 strains.Ho w ever, KpAtg5-Cerulean dots were observed only in the WT strain and ∼20% of these puncta colocalized with YFP-KpAtg8 (Fig. 5 D and  E).These r esults str ongl y suggested that KpAtg14 is necessary for recruiting the downstream ATG proteins, similar to ScAtg14 during bulk autophagy.
Atg14 is an autophagy-specific subunit of the class III phosphatidylinositol 3-kinase (PI3-K) complex 1 (hereafter referred to as PI3KC1) and is an important determinant of the PI3KC1 function r equir ed for autopha gy (Obar a and Ohsumi 2011 ).In S. cerevisiae , the PI3KC1 is composed of ScAtg14, ScAtg6, ScVps34, and ScVps15, and ScAtg14 and ScAtg6 are conjugated to each other.To investigate whether KpAtg14 interacts with KpAtg6, we constructed the str ain expr essing KpAtg14-FLAG and KpAtg6-HA and performed a coimm unopr ecipitation anal ysis.We found that KpAtg6-HA was precipitated by anti-FLAG antibodies ( Fig. S3 ), suggesting that KpAtg14 forms a complex with KpAtg6.

The C-terminal region of KpAtg14 is dispensable for autophagy
During the alignment analysis, we found that KpAtg14 and OpAtg14 had a longer C-terminal region with 100-200 amino acids than ScAtg14 and SpAtg14 ( Fig. S1 ).The structur al anal ysis by AlphaFold2 indeed indicated that KpAtg14 and OpAtg14 had a longer intrinsicall y disorder ed r egion ( Fig. S4A -D ), indicating that Atg14 of methylotrophic yeasts has a conserved unique function.Ther efor e, we constructed the Kpatg14 strain expressing a mutant KpAtg14 that lacked the C-terminal 84 amino acids (her eafter r eferr ed to as the Kpatg14-C84 str ain) ( Fig. S4E ).We monitor ed bulk degr adation in the Kpatg14-C84 str ain expr essing KpPgk1-CFP and found that the cleaved form of CFP was detected in a manner similar to the WT strain ( Fig. S4F ), indicating that the C-terminal region of KpAtg14 is not r equir ed for bulkautopha gy.We also inv estigated the pexopha gic activity in the   Kpatg14-C84 str ain expr essing KpPex11-pHluorin under macr oand micr opexopha gy-induced condition.Similar to the WT strain, the cleaved form of pHluorin was detected by immunoblot analysis under both macro-and micropexophagy-induced conditions ( Fig. S4G and H ), indicating that the C-terminal region of KpAtg14 is dispensable for autophagy.

Discussion
In this study, we established a novel FACS-based screening system to identify genes r equir ed for pexopha gy using the methylotrophic yeast K. phaffii .This method allo w ed us to sort ∼50 000 transformants at one time, which has a much higher throughput than conventional plate-based assays that scr een hundr eds of transformants (Stasyk et al. 2008 ).In addition, this novel screening method also allows the identification of ATGs r equir ed for pexophagy under macropexophagy-induced conditions.To date, micr opexopha gy-induced conditions hav e been primaril y used to identify the ATGs r equir ed for pexophagy as plate colony screening under macr opexopha gy-induced conditions has often yielded false positiv e r esults.We belie v e that the high-throughput FACSbased scr eening pr otocol for macr opexopha gy m utants established in this study will help in the identification of other novel genes necessary for pexophagy with a higher probability.
The ne wl y identified KpATG14 gene was found to be r equir ed not only for macropexophagy but also for micropexophagy (Figs 2  and 3 ) similar to the other eight ATGs that wer e discov er ed and c har acterized in pr e vious studies (Table 4 ).Our immediate goal is to discover genes required solely for either macro-or micropexophagy.As of today, ∼20 ATGs have been identified to be required for pexophagy and all of them are necessary for both macro-or micr opexopha gy pathways (Oku and Sakai 2016 ).As such, further work is needed to identify genes specific for one of the two pexophagy pathwa ys .Identification of genes r equir ed for either macro-or micropexophagy enables us to get closer to understanding the physiological significance of the use of these two distinct pathways for peroxisome degradation by yeasts.To ac hie v e this, improving the current FACS-based screening system might be an option.When combined with other techniques including gene expr ession micr oarr ays (Gallardo and Behr a 2013 ), FACS scr eenings can pr ov e to be mor e po w erful in identifying specific genes of interest.
KpAtg14 was ne wl y added to a list of genes r equir ed for pexophagy and bulk autophagy in methylotrophic yeasts.Atg14 is a k e y factor in determining the function of the PI3KC1, which is one of the core components of the autophagosome component (Obara and Ohsumi 2011 ).The role of Atg14 on autophagy and its molecular c har acteristics hav e been inv estigated in the budding yeast S. cerevisiae (Obara et al. 2006 ) and the fission yeast S. pombe (Sun et al. 2013 ), as well as in other organisms (Itakura et al. 2008, Liu et al. 2017, 2020, Thompson et al. 2021 ).Ho w e v er, a homologous protein of Atg14 has not been discov er ed in K. phaffii for so long, despite a pr e vious attempt (Farré et al. 2010 ).We belie v e that the reason for this is the longer C-terminal region that is present only in methylotrophic yeasts compared to other yeast species ( Figs S1 and S4A -D ).In mammalian Atg14, the Barkor/Atg14(L) autopha gosome-tar geting sequence (BATS) domain is conserved at the C-terminus and the domain plays a role in autophagosome targeting and membrane curvature sensing (Fan et al. 2011 ).Ho w e v er , the BA TS domain was not found to be conserved in any methylotrophic yeasts (data not shown).This could be an indication that the C-terminus of Atg14 in the methylotrophic yeasts holds a specific role.Our previous studies have shown that the Cterminal region of CbHap3, a component of the multimeric transcription factor, the CbHap complex, is r equir ed for methanolregulated gene expression, and that such a unique sequence was conserv ed among methylotr ophic yeasts, but not in S. cerevisiae (Oda et al. 2015(Oda et al. , 2016 ) ). Ho w e v er, deletion of the C-terminal r egion of KpAtg14 did not cause any effect on macr opexopha gy, micr opexopha gy or bulk autophagy in our experiments.Further analysis may be necessary to identify the specific functions of the C-terminal regions of KpAtg14 and OpAtg14.
OpAtg14 was identified by PSI-BLAST with KpAtg14, but not with ScAtg14 ( Fig. S1 ), whic h pr ompted us to further investigate whether a homologous protein of Atg14 could be found in other or ganisms.A homology searc h by PSI-BLAST with KpAtg14 identified hypothetical proteins from seven organisms, namely Candida arabinofermentans , Candida boidinii , Ogataea philodendri , Pachysolen tannophilus , Pichia kudriavzevii , Pichia membranifaciens and Wickerhamomyces pijperi (synonym Pichia pijperi ).Among the seven hypothetical proteins, those from C. arabinofermentans , P. tannophilus and W. pijperi could also be found by PSI-BLAST with Sctg14, but the others could not be, suggesting that amino acid sequences of unconventional yeasts like K. phaffii can have their homologous pr oteins in v arious yeasts , and further in vestigation could lead to a compr ehensiv e understanding of specific genes.
In summary, our novel FACS-based screening method discov er ed the pr e viousl y unidentified KpATG14 gene in the methylotr ophic yeast K. phaffii .Micr oscopic and imm unoblot analyses r e v ealed that KpAtg14 is r equir ed for both macr o-and micr opexopha gy.Our r esults indicated that KpAtg14 plays a critical role in the complete enclosure of peroxisomes in the vacuole under micr opexopha gy-induced conditions.Furthermor e, KpAtg14, like ScAtg14, recruits downstream ATG proteins and thus is required for bulk autophagic degradation.These results indicated that KpAtg14 has a conserved role in autophagic degradation among other eukaryotes.

Figure 1 .
Figure1.Establishment of the FACS-based screening system for macr opexopha gy m utants.(A) Conceptual dia gr am of the methodology adopted in the screening method for discovering macropexophagy mutants using the KpPex11-pHluorin-mCherry reporter.V, vacuole; P, peroxisome; + , pexophagy activ e; −, pexopha gy defectiv e; and FCM, flo w c ytometry.(B) Micr oscopic ima ges of WT and Kpatg30 cells expr essing KpPex11-pHluorin-mCherry.Cells were observed 6 h after the carbon-source shift from methanol to ethanol.Merged images are combined images of pHluorin and mCherry fluor escence ima ges.Bar; 2 μm.(C) Re presentati ve flo w c ytometry plot for sorting WT and atg30 cells expressing KpPex11-pHluorin-mCherry.The left panel shows the population of cells grown on methanol, whereas the right panel displays the population of cells transferred from a methanol to an ethanol-containing medium and incubated for 6 h.Scater plots of WT cells (blue) ar e ov erlaid with those of Kpatg30 cells (green).The sorting gate for scr eening macr opexopha gy m utants is indicated b y the cir cle (r ed).(D) Sc hematic ov ervie w of the experimental pr otocol.After REMI m uta genesis of WT cells expressing KpPex11-pHluorin-mCherry was introduced by transforming the pREMI-Z vector, all the transformants were collected for the first screening by FACS.The sorted candidates were further evaluated by fluorescence microscopy as the second screening.Finally, sequencing analysis was conducted to identify the mutation insertion sites and the genes required for macropexophagy.

Figure 3 .
Figure 3. Functional analysis of KpAtg14 under micropexophagy-induced conditions.(A) Schematic image and fluorescent microscopy analysis of the WT and Kpatg14 strains expressing YFP-KpAtg8 and CFP-PTS1 during micropexophagy.The vacuolar membranes were stained with FM4-64.Merged ima ges ar e combined ima ges of FM4-64, YFP-KpAtg8, and CFP-PTS1 images.DIC, differential interference contrast microscopy.Bar; 2 μm.For the sc hematic ima ge, V, Ps, VMS, and MIPA indicate v acuole, per oxisomes, VSMs, and micr opexopha gic membr ane a ppar atus, r espectiv el y. (B) Quantification of the cells with CFP diffusion in the WT and Kpatg14 str ains.Micr oscopic ima ges of the cells 2 h after micr opexopha gy induction (see Fig. 3 A) were used for analysis.Quantified values are shown as the means ± SE of three independent experiments.* * P < .01versus control.(C) Morphometric analysis of VSM formation.Quantified values are shown as the means ± SE of three independent experiments .n.s .= not significant.(D) Immunoblot analysis of pHluorin-tagged KpPex11 in K. phaffii WT, Kpatg14 , and Kpatg30 cells under micr opexopha gy-induced conditions.(E) Quantification of the intensity of (pHluorin)/(pHluorin + KpPex11-pHluorin) in the WT and Kpatg14 strains .T he bands detected 0 and 5 h after micr opexopha gy induction (see Fig. 3 D) were used for analysis.Quantified values are shown as the means ± SE of three independent experiments .n.s .= not significant and * P < .05versus control.(F) Fluorescent microscopy analysis of the WT strain expressing YFP-KpAtg8 and KpAtg14-Cerulean during micr opexopha gy.The v acuolar membr anes wer e stained with FM4-64.Mer ged ima ges ar e combined ima ges of FM4-64, YFP-KpAtg8, and KpAtg14-Cerulean images.DIC, differential interference contrast microscopy.Bar; 2 μm.Arrows indicate the colocalization of YFP-KpAtg8 and KpAtg14-Cerulean.

Figure 4 .
Figure 4. Functional analysis of KpAtg14 under starvation-induced mitophagy conditions.(A) Immunoblot analysis of YFP-tagged KpIdh1 in K. phaffii WT and Kpatg14 cells under nitr ogen-starv ation conditions.(B) Quantification of the intensity of (YFP)/(YFP + KpIdh1-YFP) in the WT and Kpatg14 strains .T he bands detected 0 and 5 h after mitophagy induction (see Fig. 4 A) were used for anal ysis.Quantified v alues ar e shown as the means ± SE of three independent experiments .n.s .= not significant.

Figure 5 .
Figure 5. Functional analysis of KpAtg14 under bulk-autophagy-induced conditions.(A) Immunoblot analysis of CFP-tagged KpPgk1 in K. phaffii WT and Kpatg14 cells under bulk autophagy-induced conditions.(B) Quantification of the intensity of (CFP)/(CFP + KpPgk1-CFP) in the WT and Kpatg14 strains .T he bands detected 0 and 5 h after bulk-autophagy induction (see Fig. 5 A) were used for analysis.Quantified values are shown as the means ± SE of three independent experiments .n.s .= not significant and * * P < .01versus control.(C) Fluorescent microscopy analysis of the WT strain expressing YFP-KpAtg8 and KpAtg14-Cerulean during bulk autophagy.The vacuolar membranes were stained with FM4-64.Merged images are combined images of FM4-64, YFP-KpAtg8, and KpAtg14-Cerulean ima ges.DIC, differ ential interfer ence contr ast micr oscopy.Bar; 2 μm.Arr ows indicate the colocalization of YFP-KpAtg8 and KpAtg14-Cerulean.(D) Fluorescent microscopy analysis during bulk-autophagy.The vacuoles were stained with FM4-64.To visualize de novo -synthesized PAS and downstream factor, YFP-KpAtg8 and KpAtg5-Cerulean were used as their marker.The vacuolar membr anes wer e stained with FM4-64.Mer ged ima ges ar e combined ima ges of FM4-64, YFP-KpAtg8 and KpAtg5-Cerulean ima ges.DIC, differential interfer ence contr ast micr oscopy.Bar; 2 μm.Arrows indicate the colocalization of YFP-KpAtg8 and KpAtg5-Cerulean.(E) Ratio of KpAtg5 puncta colocalizing with KpAtg8, estimated from analysis of the fluorescent images shown in Fig. 5 (D).For each sample, a minimum of 50 cells were analyzed.Quantified values are shown as the means ± SE of three independent experiments.* * P < .01versus control in each condition.

Table 3 .
Plasmids used in this study.

Table 4 .
List of ATGs identified in this study.